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Research Article
Rapsyn mediates subsynaptic anchoring of PKA type I
and stabilisation of acetylcholine receptor in vivo
Kyeong-Rok Choi1, Marco Berrera2, Markus Reischl3, Siegfried Strack1, Marina Albrizio1, Ira V. Röder1,
Anika Wagner1, Yvonne Petersen1, Mathias Hafner4, Manuela Zaccolo2 and Rüdiger Rudolf1,*
1
Institut für Toxikologie und Genetik, and 3Institut für Angewandte Informatik, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1,
76344, Eggenstein-Leopoldshafen, Germany
2
Institute of Neuroscience and Psychology, University of Glasgow, Glasgow G12 8QQ, UK
4
Institut für Molekular- und Zellbiologie, Hochschule Mannheim, Paul-Wittsack-Strasse 10, 68163, Mannheim, Germany
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 22 September 2011
Journal of Cell Science 125, 714–723
ß 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.092361
Summary
The stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative
action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain
where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12
and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation
and bimolecular fluorescence complementation approaches identify an a-helical stretch of rapsyn to be crucial for binding to the
dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn a-helical
sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking
the a-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that
rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse
maintenance.
Key words: Neurotransmitter receptor recycling, A-kinase anchoring protein, Rapsyn, Skeletal muscle
Introduction
Metabolic stabilisation of acetylcholine receptors (AChRs) at the
neuromuscular junction (NMJ) is dependent on muscle activity
(Lomo and Rosenthal, 1972; Stanley and Drachman, 1981) as well
as on postsynaptic signalling (Fambrough, 1979; Nelson et al.,
2003; Shyng et al., 1991; Witzemann et al., 1987; Xu and Salpeter,
1997; Xu and Salpeter, 1999) and protein transport processes
(Yampolsky et al., 2010b). Particularly, receptor recycling plays an
important role in the activity-dependent regulation of AChR
turnover (Akaaboune et al., 1999; Bruneau et al., 2005; Röder et al.,
2008). To mediate efficient AChR recycling, the subsynaptic
enrichment of a complex of AChR, myosin Va, and protein kinase
A (PKA) type I was found to be essential (Röder et al., 2010;
Rudolf et al., 2011; Yampolsky et al., 2010a). This fits with a longproposed function of PKA type I in AChR stabilisation (Nelson
et al., 2003; Shyng et al., 1991; Xu and Salpeter, 1997), which is
probably mediated by neurogenic factors, such as a-calcitonin
gene-related peptide (Nelson et al., 2003).
A missing link in this scenario has been the factor that
mediates accumulation of PKA type I in proximity to the NMJ.
Typically, PKAs are targeted to specific subcellular sites by
means of A-kinase anchoring proteins (AKAPs) (Colledge
and Scott, 1999). These are structurally heterogeneous proteins,
which exhibit subcellular targeting sequences and PKA binding
sites. In addition, most AKAPs also act as scaffold proteins by
binding further factors, such as receptors, phosphatases etc. By
that means, AKAPs act as signalling integrators that link PKA,
effector molecules and signal modifiers in close proximity to
the expected site of signal arrival (Colledge and Scott,
1999). Together with other mechanisms, this allows the cell to
efficiently compartmentalise different cAMP-dependent processes
and to maintain high signal specificity (Zaccolo, 2009). In most
cases, AKAPs mediate binding to PKA regulatory subunits (PKAR) through amphipathic a-helical sequences, which interact with
an N-terminal stretch on PKA-R called the dimerisation and
docking domain (DD domain) (Colledge and Scott, 1999). DD
domains are specific for the different PKA-Rs known in
mammals.
The receptor-associated protein of the synapse (rapsyn, also
known as RAPSN) is a 43 kDa protein, which is strongly
associated with AChRs at the NMJ and plays an important role
in clustering of AChRs at the synapse (Frail et al., 1988; Gautam
et al., 1995). From N- to C-terminus, it consists of a myristoylation
site, seven tetratricopeptide repeats, an amphipatic a-helical
stretch, a RING H2 domain and consensus sequences for PKC
and PKA phosphorylation (Ramarao et al., 2001; Ramarao and
Cohen, 1998). So far, the a-helical stretch (residues 299–331) has
been described as important for AChR clustering (Ramarao et al.,
2001; Ramarao and Cohen, 1998). In the light of our recent finding
that a complex forms between recycling AChRs, myosin Va and
PKA type I, we have investigated whether rapsyn is responsible for
PKA localisation at the NMJ.
Rapsyn in PKA anchoring and NMJ plasticity
Results
Journal of Cell Science
In silico modelling predicts interaction between rapsyn
amphipathic helix and PKA-RIa DD domains
Previously, rapsyn residues 298–331 were described as forming a
coiled-coil domain including an amphipathic helix (Ramarao
et al., 2001). Amphipathic helices are known to mediate
interaction of AKAPs with PKA (for a review, see Colledge
and Scott, 1999). In particular, the specificity determinants of
AKAP10 binding to PKA-RIa (also known as PKRAR1A) were
recently elucidated by the X-ray structure of the complex
between the amphipathic helix and DD domains (Sarma et al.,
2010). AKAP10 helix bound diagonally to the PKA-RIa DD
domain homodimer. Four hydrophobic interaction pockets were
characterised and they involved AKAP10 residues from the
hydrophobic face of the amphipathic helix equally spaced on the
sequence. Previously, the profile Hidden-Markov model (HMM)
of the AKAP amphipathic helix motif that binds to PKA has been
successfully employed to predict novel PKA anchoring proteins
despite their low sequence identity (McLaughlin et al., 2011). We
have used this approach to locate the PKA binding motif of
rapsyn by alignment of its sequence to the profile HMM that is
calculated from the alignment of 22 different PKA-binding
AKAP sequences (McLaughlin et al., 2011) (Fig. 1A).
Under the assumption that rapsyn interacts with PKA in an
AKAP-like manner, to characterise the interactions stabilising the
complex we then modelled the complex between the rapsyn
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coiled-coil fragment and PKA-RIa on the basis of the X-ray
structure of AKAP10 amphipathic helix bound to DD domains of
PKA-RIa (Sarma et al., 2010) and of the corresponding sequence
alignment (Fig. 1A). Due to missing template structures for
residues 298–304, we modelled only rapsyn residues 305–331 as
a-helix. Then, the complex was immersed in a water solvent box
and 100 nanoseconds of molecular dynamics (MD) simulation
under standard conditions were performed to test the stability of
the complex and of the protein–protein interactions. Root square
mean deviation (RMSD) fluctuated at about 0.2 nm and no
conformational change or repositioning of the helix were
observed within the investigated time frame. Interactions
between PKA-RIa and rapsyn were mainly hydrophobic
contacts between residues at the dimerisation interface of
DD domains and residues from the hydrophobic face of the
amphipatic helix, similar to those characterised in detail for
AKAP10 (Sarma et al., 2010). Salt bridge interactions were
observed between the lysine 311 of rapsyn (rapsyn-K311) and
PKA-RIa-E17 as well as between rapsyn-K327 and PKARIa-E1799 (on the opposite DD domain helix). Hydrophobic
interactions were estimated for each residue on the basis of a
contact surface calculation and averaged throughout the
MD trajectory. Rapsyn residues with large contact surface
(.0.40 nm2) were A308, K311, A312, L315, A316, V319,
L323 and K327 (Fig. 1B). PKA-RIa residues forming a large
surface of contact with rapsyn were L13, E17, Q26, K30, I33 and
Fig. 1. In silico structural model of the complex between
rapsyn a-helical domain and DD domain homodimer of PKARIa. (A) The sequence of rapsyn amphipathic helix (residues
305–331) was aligned with the AKAP10 amphipathic helix
(residues 628–654) via profile HMM calculated from the
sequence alignment from McLaughlin (McLaughlin et al., 2011).
The sequence alignment of rapsyn with several dual-specificity
AKAPs is shown. The residues lining the four interaction pockets
as defined by Sarma and colleagues (Sarma et al., 2010) are
boxed. Rapsyn residues forming large (.0.40 nm2) surface of
contact with PKA-RIa DD domain homodimer, averaged over the
MD trajectory, are underlined. (B) Surface of rapsyn residues in
contact with PKA-RIa DD domain homodimer. Residues with
large contact surface are underlined. (C) Cartoon of the complex
in water solution as determined by in silico modelling. Rapsyn
helix and residues are coloured in red, DD subunits and residues
are coloured in brown and silver. Residues forming large contact
surface are drawn in balls and sticks and coloured by atom type.
In addition, side chains of rapsyn residues are emphasised by a
red surface. Inset: Helical wheel diagram of rapsyn amphipathic
helix. Residues that form large surface of contact with DD
domain homodimer are boxed.
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Journal of Cell Science 125 (3)
V34 on one partner of the DD domain dimer and residues L139,
Q269, and K309 on the opposite side of the DD domain (Fig. 1C).
The inset in Fig. 1C shows the rapsyn residues on the helix that
face the PKA in boxes.
Journal of Cell Science
Rapsyn interacts with PKA type I in cells
On the basis of these findings, we assessed whether rapsyn
interacts with PKA type I in cells. To this end we
immunoprecipitated rapsyn from lysates of differentiated
C2C12 myoblast cells. On western blots, we then confirmed
the co-precipitation of PKA-RIa (Fig. 2A). By contrast, aactinin, a negative control, did not precipitate under these
conditions (Fig. 2A). To look at whether the amphipathic helix
domain of rapsyn is important for the interaction with PKA-RIa,
we performed immunoprecipitation using PKA-RIa-specific
antibody on lysates produced from human embryonic kidney
cell-derived T-REx293 cells transfected with either full-length
rapsyn or rapsyn lacking the amphipathic helix (rapsynDH,
see Fig. 2B for molecular schemes). As depicted in Fig. 2C,
full-length rapsyn co-precipitated with PKA-RIa. Conversely,
rapsynDH could not be detected in the precipitate, showing that
the amphipathic helix of rapsyn is essential for the interaction
with PKA type I.
Next, we studied the interaction of rapsyn with PKA type I by
means of bimolecular fluorescence complementation (BiFC)
assays (Hu et al., 2002). T-REx293 cells were co-transfected with
mCherry as a transfection marker and different combinations
of BiFC constructs (see supplementary material Fig. S1 for
molecular schemes). In some cases, mCherry was preceded by
the RI disruptor peptide RIAD (RIAD-mCherry) (Carlson et al.,
2006). In addition to the N- (VN) or C-terminus (VC) of venus
protein, BiFC constructs contained either full-length rapsyn and
PKA-RIa or truncated versions of these proteins (rapsynDH and
Ddd-PKA-RIa). Two days after transfection, cells were fixed
and nuclei stained with draq5. Confocal imaging was used to
determine the presence of BiFC (Fig. 3A–D). Quantitative
analysis (Fig. 3E) showed that about 40–50% of mCherrypositive cells exhibited punctiform BiFC signals upon cotransfection of full-length rapsyn and PKA-RIa. The amount of
BiFC-positive cells significantly dropped to 10–30% in the
presence of RIAD-mCherry and the decrease was even more
prominent upon coexpression of mutant rapsyn or mutant PKARIa. Results were similar irrespective of whether rapsyn and
its mutants were present in VC fusions and PKA-RIa and its
mutants in VN fusions (Fig. 3E, left half) or in the reverse
orientation (Fig. 3E, right half). These results corroborate the
immunoprecipitation data and indicate an interaction between
rapsyn and PKA-RIa, which relies on the DD domain of PKARIa and the a-helical stretch of rapsyn.
In vivo BiFC detects interaction of rapsyn and PKA type I at
the NMJ
To verify whether the interaction between rapsyn and PKA-RIa
also occurs in vivo and to gauge its localisation, we performed
BiFC assays in the muscles of living mice. BiFC constructs were
co-transfected in different combinations in mouse tibialis anterior
muscles. Ten days later, a-bungarotoxin (BGT), a marker for
AChRs, coupled to the fluorescent dye Alexa Fluor 647 (BGTAF647) was injected to label NMJs. The muscles were then
monitored using in vivo imaging. Upon coexpression of rapsynVN and PKA-RIa-VC, a high enrichment of BiFC signals was
detected in punctiform structures almost exclusively in close
proximity to the NMJ (Fig. 4, top row; for low power images see
supplementary material Fig. S2). A very similar distribution was
Fig. 2. Rapsyn co-precipitates with PKA-RIa. (A) After 10 days
of differentiation, C2C12 cells were harvested, and lysates
prepared and incubated in the presence or absence of rapsyn
antibody (AB). After immunoprecipitation, eluates were run on
SDS-PAGE and then probed on western blot using antibodies
against rapsyn, PKA-RIa and a-actinin (negative control). The
bands show western blot signals in lysates and eluates. (B,C) TREx293 cells were transfected with HA-tagged versions of either
rapsyn (rapsyn-HA) or a rapsyn mutant lacking the a-helical
domain (amino acids 299–331; rapsynDH-HA). Lysates were
prepared, immunoprecipitated with monoclonal anti-PKA-RIa
antibody and subsequently probed on western blots for HA and
PKA-RIa. (B) Schemes of transfected fusion constructs.
(C) Western blot signals in lysates and eluates. Note that only fulllength rapsyn co-precipitates with PKA-RIa. The graph on the
right shows a densitometric analysis. Depicted are mean 6 s.e.m.
(n53 experiments) of the eluate band intensities normalised to the
lysate. **P,0.01 according to Welch test.
Rapsyn in PKA anchoring and NMJ plasticity
Journal of Cell Science
shown by coexpression of PKA-RIa-VN and PKA-RIa-VC
(Fig. 4, middle row) and was also described previously for fulllength PKA-RIa and fluorescent proteins fused to the PKARIa DD domain (Barradeau et al., 2001; Röder et al., 2010).
Conversely, co-transfection of rapsyn-VN and rapsyn-VC led to
the typical rapsyn-like staining, which is almost completely in the
NMJ (Fig. 4, bottom row). Strong rapsyn–rapsyn-BiFC signals
were expected due to the well-known self-interacting properties
of rapsyn (Ramarao et al., 2001; Ramarao and Cohen, 1998).
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This shows that there is specific interaction between PKA-RIa
and rapsyn in close proximity to the NMJ.
Expression of the rapsyn a-helical peptide delocalises
PKA type I from its subsynaptic microdomain
Next, we investigated whether the amphipathic helix of rapsyn
might function as an AKAP-RI disruptor peptide in vivo.
Therefore, a construct expressing this amino acid sequence
(rapsyn aHelix) was expressed in the tibialis anterior muscle of
live mice. As a control, contra-lateral muscles were transfected
with a control peptide in which a number of residues were
mutated. Ten days after transfection, muscles were extracted,
sliced and the NMJs stained with BGT-AF647. In addition, these
slices were immunostained against PKA-RI or rapsyn and then
analysed using confocal microscopy (Fig. 5). Notably, although
rapsyn staining was equal in controls and rapsyn aHelixtransfected muscles (Fig. 5C,D), PKA-RI staining was clearly
reduced in the NMJ region upon expression of the rapsyn aHelix
(Fig. 5A,B). Quantitative analysis showed that in controls and
rapsyn aHelix-transfected muscles 95.760.1% and 97.060.7%,
respectively, were rapsyn-positive (n53 different muscles, at
least 120 NMJs analysed per condition). For PKA-RI signals,
the values were significantly different with 71.966.3% and
39.768.6% of positive fibres (n53 different muscles, at least 120
NMJs analysed per condition; P50.022 according to Welch test).
To further consolidate the hypothesis that rapsyn aHelix
peptide delocalises PKA-RIa from its subsynaptic site, we
used a co-transfection approach (Fig. 5E,F). Muscles were cotransfected with the PKA-RIa localisation marker, RIa-EPAC,
and either rapsyn aHelix or the control peptide. Ten days later,
NMJs were labelled with BGT-AF647 and then imaged using in
vivo confocal microscopy (Fig. 5E). On average, the amount of
NMJs exhibiting RIa-EPAC accumulations in the subsynaptic
region decreased from 73.463.7% (n54 mice) in the control
to 21.463.2% (n54 mice) in the presence of rapsyn aHelix
(Fig. 5F). These results strongly indicate that rapsyn is indeed an
AKAP targeting PKA-RIa to the NMJ region.
Rapsyn a-helical peptide induces high AChR turnover and
synaptic fragmentation
Finally, we asked whether the presence of the rapsyn peptide
could mimic the effects of the PKA-RI-specific AKAP disruptor
peptide, RIAD (Röder et al., 2010). This was shown to release
PKA-RI from its subsynaptic domain and to induce an increased
AChR turnover as well as fragmentation of NMJs into numerous
separate gutters. Thus, as before, mouse tibialis anterior muscles
Fig. 3. BiFC shows interaction between rapsyn and PKA-RIa. T-REx293
cells were co-transfected with mCherry or RIAD–mCherry and different
combinations of BiFC constructs harboring N- (VN) or C-terminals (VC) of
Venus and an HA tag together with rapsyn, rapsynDH, PKA-RIa or DddPKA-RIa (for schemes see supplementary material Fig. S1). Then, cells were
fixed, stained with draq5, a nuclear marker, and imaged using confocal
microscopy. Subsequently, the fraction of mCherry- or RIAD–mCherrypositive cells showing BiFC signals was determined. (A,B) Fields of cells cotransfected with PKA-RIa-VN and rapsyn-VC (A) or with PKA-RIa-VN and
rapsynDH-VC (B). Overlays show BiFC signals (green), mCherry signals
(red) and nuclei (blue). Scale bars: 50 mm. (C,D) Blow-ups of regions in A
and B. Scale bars: 10 mm. (E) Quantification of BiFC-positive cells showing
mean 6 s.e.m. (n$3 independent experiments). *P,0.05, **P,0.01
according to Welch test.
Journal of Cell Science 125 (3)
Journal of Cell Science
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Fig. 4. In vivo BiFC shows interaction between rapsyn and PKA-RIa in a
subsynaptic region in live mouse muscle. Mouse tibialis anterior muscles
were co-transfected in different combinations as indicated with BiFC
constructs harboring N- (VN) or C-terminal (VC-) fragments of Venus protein
fused to rapsyn or PKA-RIa. After 10 days, muscles were imaged using in
vivo confocal microscopy. NMJs were marked with BGT-AF647. Panels
depict maximum z-projections of BiFC (green), BGT-AF647 (red) signals and
overlays of both (yellow if colocalising) from about 20 confocal slices taken
at 1.5 mm interslice interval. Scale bar: 40 mm.
were transfected with rapsyn aHelix and contra-laterally with the
control peptide. Simultaneously, AChRs present at this time
point were labelled with infrared fluorescent BGT-AF647 (‘old
receptors’). Ten days later, red fluorescent BGT-AF555 was
applied to mark newly formed AChRs (‘new receptors’). Then,
muscles were monitored by in vivo imaging and NMJs were
segmented and analysed as described previously (Fig. 6A)
(Röder et al., 2010). Notably, in the muscles expressing rapsyn
aHelix, the fraction of ‘new receptors’ within the total population
of labelled AChRs was highly enriched compared with the
control (Fig. 6A). Quantitative analysis revealed values of
0.5960.13 and 0.0660.04 for rapsyn aHelix-transfected and
control muscles, respectively (n56 muscles and at least 150
NMJs were analysed for each condition; P,0.01 according to
Welch test) (Fig. 6B). Also, rapsyn aHelix induced significantly
more fragmentation of NMJs (supplementary material Fig. S3).
On average, 4.260.8 and 2.160.3 fragments per NMJ were
measured in the presence and absence of the rapsyn aHelix,
respectively (n56 muscles P,0.05 according to Welch test)
(Fig. 6B). Together, these data show that binding of PKA to
rapsyn is crucial for AChR stability and NMJ integrity and that
the rapsyn aHelix peptide reproduces the same phenotype as
previously observed upon expression of the PKA-RI-specific
AKAP disruptor peptide, RIAD (Röder et al., 2010).
To further substantiate the role of rapsyn in stablising AChRs,
we performed two more experiments. First, we tested whether the
rapsyn mutant lacking the amphipathic helix (rapsynDH) would
act in a dominant-negative manner. Therefore, tibialis anterior
muscles were transfected with this construct and, as before, old
and new AChR pools were labelled at a temporal distance
of 10 days with BGT-AF647 and BGT-AF555, respectively.
Subsequent in vivo imaging (Fig. 6A) and automated quantitative
data analysis (Fig. 6B) showed that rapsynDH was as effective in
destabilising AChRs as the rapsyn aHelix peptide (‘new
receptor’/total receptor signals, 0.5960.14, n56 muscles, 210
NMJs analysed). Also the amount of fragments per synapse
(5.1160.46, n56 muscles, 210 NMJs analysed) was increased in
a highly significant manner as compared with the control.
Second, we used a recently established radiochemical assay
(Strack et al., 2011) to determine the AChR half-lives in the
presence or absence of rapsyn aHelix peptide. Therefore, 1 week
after transfection (left, control; right, aHelix peptide) muscles
were pulse-labelled with 125I-BGT. Then, AChR turnover was
measured over the next 4 weeks. Although in both sides AChRs
displayed the typical two-term lifetime distribution with a shortlived (half-life, t1/2 1.1 days) and a long-lived population of
receptors, AChR lifetime was clearly affected in the rapsyn
aHelix peptide-expressing muscles (Fig. 6C). In particular, the
short-lived receptors pool increased from 17.263.1% (n55 mice)
in the control to 35.362.8% (n55 mice) in the rapsyn aHelixexpressing muscles. These data corroborate the imaging data and
clearly indicate that the rapsyn aHelix peptide strongly impacts
on AChR lifetime.
Discussion
Rapsyn is known to be important for the formation and
maintenance of the vertebrate NMJ (Gautam et al., 1995).
There is a wealth of studies showing a direct correlation between
the amount of rapsyn present at NMJs and the abundance or the
metabolic stability of synaptic AChRs (Brockhausen et al., 2008;
Gervasio et al., 2007; Gervasio and Phillips, 2005; Luo et al.,
2008; Martinez-Martinez et al., 2009; Wang et al., 1999).
Furthermore, mutations in the RAPSN gene are amongst the
most frequent causes for myasthenic syndromes, which are
characterised by activity-dependent muscle weakness (Beeson
et al., 2008; Müller et al., 2007). Together, these findings place
rapsyn in the centre of regulatory processes that mediate
stabilisation of synaptic AChRs.
Given previous reports highlighting a role of cAMP and PKA
type I in AChR stabilisation (Nelson et al., 2003; Shyng et al.,
1991; Xu and Salpeter, 1997) and in the light of our previous
finding that a complex of recycling AChRs, myosin Va, and
PKA-RI is crucial for the stabilisation of AChRs (Röder et al.,
2010), it was important to establish which protein links PKA type
Rapsyn in PKA anchoring and NMJ plasticity
Journal of Cell Science
I to the subsynaptic microdomain. Prime players in targeting
PKA molecules to subcellular sites are AKAPs, and one of their
hallmark features is a PKA-R-interacting amphipathic a-helical
stretch (Colledge and Scott, 1999). Owing to the presence of
such a domain, its described role in AChR stabilisation and its
Fig. 5. See next page for legend.
719
molecular interaction with AChRs, rapsyn appears to be an ideal
candidate for mediating the anchoring of PKA-RI close to the
NMJ. In this study we provide biochemical, in vitro, in silico and
in vivo data that strongly support the role of rapsyn as the AKAP
that mediates accumulation of PKA type I close to the NMJ,
Journal of Cell Science
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Journal of Cell Science 125 (3)
which in turn is a pre-requisite for proper stabilisation of synaptic
AChRs.
We first compared rapsyn amphipathic helix (Ramarao et al.,
2001) to a sequence alignment of 22 known AKAP-binding
proteins (McLaughlin et al., 2011), which includes also AKAP10.
The structure of AKAP10 amphipathic helix in complex with
PKA-RIa was recently solved (Sarma et al., 2010) and was used
here as template to model the rapsyn–PKA-RIa complex. We
found that the AKAP10 residues that occupy the four main
interaction pockets as defined by Sarma and colleagues (Sarma
et al., 2010) are substituted in rapsyn by similar hydrophobic
residues, except for K311 and S324. These two residues are
partially solvent-exposed and the corresponding alignment
positions are not conserved. However, K311 aligns with lysine
from AKAP12 and MTG16, and S324 aligns with other polar
residues (Fig. 1A). Besides the residues from the four interaction
pockets, A308 and K327 from the N- and C-termini of the helix,
respectively, were found to contribute to the binding stability by
forming additional contacts with the PKA-RIa DD domain
homodimer (Fig. 1C). The network of interprotein interactions
overlaps with the AKAP10–PKA-RIa interaction pattern
observed in the X-ray structure of the complex (Sarma et al.,
2010) and suggests that rapsyn interaction with PKA-RIa is
mostly non-polar. Nevertheless, we cannot exclude the possibility
that regions at the N-terminal of the rapsyn amphipathic helix
(not included in our model) could enhance targeting to PKA-RIa
by forming additional interactions, as proposed for dualspecificity AKAPs (Jarnaess et al., 2008).
Biochemical pull-down (Fig. 2) and BiFC experiments (Fig. 3)
obtained in C2C12 and T-REx293 cells corroborate the
modelling data and show that PKA type I and rapsyn interact
with each other by binding of the PKA-RI DD domain to the
rapsyn a-helical domain. In vivo BiFC, using PKA-RIa and
rapsyn as interaction partners showed a strong enrichment of
BiFC signals in punctiform structures close to the NMJ but not
Fig. 5. Peptide encompassing rapsyn a-helical domain displaces PKARIa from its subsynaptic microdomain in vivo. (A–F) A peptide
encompassing the rapsyn a-helix (rapsyn aHelix) or a mutant control peptide
(control) was transfected (A–D) or co-transfected together with RIa-EPAC
(E,F), a marker for the localisation of PKA-RIa, into mouse tibialis anterior
muscles. After 10 days, muscles were extracted, sliced and stained against
AChR with BGT-AF647, and with antibodies against PKA-RI (A,B) or
rapsyn (C,D), or injected with BGT-AF647 and then imaged in vivo (E,F).
Stained sections were analysed with confocal microscopy, and antibody
staining intensities in the NMJs were determined (see Materials and Methods
section for details). (A,C) Confocal slices of muscles transfected as indicated.
In overlays, antibody signals are shown in green, BGT signals in red. Scale
bar: 50 mm. (B,D) Cumulative plots showing the antibody staining intensities
in all analysed NMJs. All NMJs right of the zero value (dotted line) were
counted as PKA-RI- or rapsyn-positive. (E) Images depict maximum zprojections of 80 (upper panels) and 97 optical slices (lower panels) taken at
3 mm interslice intervals of muscles co-transfected as indicated on the left.
Images show BGT (AChR), RIa-EPAC fluorescence signals or pseudocoloured NMJ outlines. In the outlines: NMJs in white, synapses of RIaEPAC-negative fibres; NMJs in yellow, synapses of RIa-EPAC-positive
fibres with subsynaptic RIa-EPAC accumulations; NMJs in red, synapses of
RIa-EPAC-positive fibres without subsynaptic RIa-EPAC accumulations.
Scale bar: 200 mm. (F) Quantitative analysis showing the average amount
(mean 6 s.e.m.) of NMJs of RIa-EPAC-positive fibres with subsynaptic RIaEPAC accumulations. n54 mice; 382 NMJs (control) and 425 NMJs (aHelix)
were analysed. **P,0.01 according to Welch test.
within the NMJ (Fig. 4). Notably, no or very few BiFC signals
were detected only a few micrometers away from the NMJ
(supplementary material Fig. S2). This pattern was very similar
to that of PKA-RIa and PKA-RIa-BiFC signals. Conversely,
rapsyn–rapsyn BiFC signals almost perfectly matched the NMJ
staining and showed only few punctuate structures. To explain
the discrepancy between the distributions of the rapsyn–PKA
BiFC and the rapsyn–rapsyn BiFC, it is important to consider
two main factors: first, a small pool of rapsyn is known to
be constantly present in intracellular, vesicular compartments
although it is primarily found in the NMJ (Bruneau and
Akaaboune, 2010; Gervasio and Phillips, 2005). Second, BiFC
between two interaction partners occurs at their site of contact
(Hu and Kerppola, 2003) and therefore indicates the location of
interaction rather than the steady-state distribution of both
proteins. Based on our findings and the general aspects of
rapsyn and BiFC as just mentioned, we speculate that rapsyn
interacts with PKA-RIa in a domain close to the NMJ. As shown
recently, the accumulation of PKA-RIa in this region is crucial
for proper AChR stabilisation (Röder et al., 2010; Röder et al.,
2008; Yampolsky et al., 2010a).
In previous reports, the rapsyn a-helical domain was described as
mediating AChR clustering at the surface of human embryonic
kidney cells coexpressing different rapsyn mutants plus all AChR
subunits (Ramarao et al., 2001; Ramarao and Cohen, 1998).
Essentially, in these studies BGT-positive patches on the cell surface
were present or absent upon coexpression of wild-type rapsyn or
rapsyn lacking the a-helical domain, respectively. Although these
results nicely showed the importance of the a-helical domain for
AChR surface exposure, it remained unclear how rapsyn mediates
this process. Although our study does not rule out a direct AChR
clustering effect of the rapsyn a-helical domain, one might also
envisage an indirect mechanism by which rapsyn, acting as an
AKAP, might enhance PKA-dependent effector phosphorylation
and enable subsequent AChR clustering. Because the mechanisms
of AChR clustering and stabilisation are still elusive, this question
has to remain open for the moment.
We designed peptides mimicking the rapsyn a-helical domain
to test their functional effect as potential AKAP disruptor
peptides in the living mouse. These peptides, but not mutated
control peptides, efficiently delocalised PKA-RI from the NMJ
(Fig. 5) and induced significant alterations in AChR turnover and
NMJ morphology (Fig. 6). These effects remarkably resembled
those induced by the PKA-RI-specific AKAP-disruptor peptide,
RIAD (Carlson et al., 2006), on the same parameters in mouse
NMJs (Röder et al., 2010). Overexpression of a rapsyn deletion
mutant lacking the amphipathic helix had a similar effect on
AChR turnover and NMJ morphology, which strongly suggests
that rapsyn is an AKAP for PKA type I in the NMJ and further
corroborates the important role of cAMP–PKA-dependent
signalling in regulating AChR turnover and NMJ morphology.
In extension to our previous model, a small pool of extrasynaptic
rapsyn might therefore anchor PKA-RIa to the AChR recycling
compartment. Our data also fit the observation of a high turnover
rate of rapsyn at the NMJ (Bruneau and Akaaboune, 2010),
because our model would benefit from a reversible and nonpermanent interaction between rapsyn and AChR.
We envisage that rapsyn recycles with AChRs by some kind of
membrane transport. During its presence in a putative recycling
compartment, some post-translational modification of rapsyn
might enable its transient binding of PKA-RIa. This would allow
Rapsyn in PKA anchoring and NMJ plasticity
721
Journal of Cell Science
Fig. 6. Peptide encompassing rapsyn a-helical
domain and rapsyn-DHelix alter AChR turnover and
fragmentation of NMJs in live mouse muscle.
(A,B) A peptide encompassing the rapsyn a-helix
(rapsyn aHelix), a mutant control peptide (control) or
rapsyn-DHelix was transfected into mouse tibialis
anterior muscles. AChRs present on the cell surface at
the time point of transfection were labelled by locally
injected BGT-AF647 (‘old receptors’). After 10 days,
muscles were injected with BGT-AF555 to mark
AChRs now available (‘new receptors’). Then, muscles
were imaged by in vivo confocal microscopy and
automatically segmented and analysed. (A) Maximum
z-projections of automatically segmented confocal slices
taken at an interslice interval of 1.5 mm. In overlays,
‘old receptors’ are shown in green and ‘new receptors’
in red. Scale bar: 50 mm. (B) Quantitative analysis of
AChR turnover and NMJ fragmentation. Shown are
average values (mean 6 s.e.m.) of the fractions of all
pixels in NMJs with dominant ‘new receptor’ signals as
a function of the numbers of fragments per NMJ (n56
muscles). (C) Seven days after transfection, muscles
were injected with 125I-BGT to pulse-label AChRs.
Then, residual 125I-activity was measured at depicted
time intervals. The time point of pulse labelling is t0.
The graph shows the residual 125I-emission in the
injected hindlimbs as a function of time after pulse
labelling. Dots represent measured values
(mean 6 s.e.m., n55 mice) and lines indicate two-term
exponential fits. Data are normalised to the mean values
measured on day 1. *P,0.05, **P,0.01 according to
Welch test.
enrichment of PKA-RIa close to the NMJ. Upon arrival of
an appropriate local cAMP stimulus, PKA-RIa might then
phosphorylate effector proteins leading to the stabilisation of
synaptic AChRs. It remains a major future task to unravel how
such receptor stabilisation is mechanistically performed.
Materials and Methods
Chemicals, antibodies and cDNAs
Primary antibodies were purchased against a-actinin (Sigma-Aldrich, Hamburg,
Germany), haemagglutinin (HA; Sigma-Aldrich), GAPDH (Santa Cruz
Biotechnology, Heidelberg, Germany), PKA-RIa (BD Bioscience, Heidelberg,
Germany) and rapsyn (Lifespan Bioscience, Seattle, WA), and secondary antibodies
conjugated to horseradish peroxidase (Dako, Hamburg, Germany), Alexa Fluor 488
or Alexa Fluor 546 (Invitrogen, Darmstadt, Germany). BGT-AF555 and BGTAF647 were from Invitrogen. Rapsyn-HA and DH-rapsyn-HA vector constructs for
co-precipitation used wild-type (1,236 bps, 412 amino acids) and a a-helical coiledcoil domain deletion mutant (deleted sequence: bps 892–996 corresponding to
amino acids 298–332) of rapsyn cDNAs. They were amplified by PCR from a
rapsyn–GFP expression vector (Jonathan Cohen, Harvard Medical School, Boston,
MA) with primers containing a C-terminal HA tag and were then subcloned into
BamHI and EcoRI cleaved pcDNA3.1(+) (Invitrogen). For BiFC, wild-type (1,143
bps, 381 amino acids) and a DD domain deletion mutant (Ddd-PKA-RIa; lacking
bps 1–186 corresponding to amino acids 1–62) of PKA-RIa cDNAs were amplified
by PCR from PKA-RIa expression vector. Wild-type and DH-mutant of rapsyn
(without HA tag) were prepared as described above. Amplified cDNAs were cloned
into pBiFC-VN or pBiFC-VC vectors containing a HA tag as described (Röder et al.,
2010). The amino acid sequence GSGGGGGSG was used as a linker between target
molecule and Venus fragment. HA tags were positioned at N- and C-terminal of
rapsyn and PKA-RIa, respectively. For functional studies, the cDNA sequences of
wild-type (rapsyn aHelix; amino acids 280–331) and mutant (rapsyn aHelix mut;
N281, G284, Q285, H287, V288, L289, L306, I309, Q313, E317 substituted to
alanine) a-helical coiled-coil domain of rapsyn were amplified by PCR and cloned
into EcoRI and XhoI cleaved pcDNA3.1(+) (Invitrogen).
Cells, animals and transfection
For culture cell transfection, T-REx293 cells (Invitrogen) were seeded on 10-cm
petri-dishes (Greiner Bio-One, Frickenhausen, Germany) and cultured in 10% FBS
722
Journal of Cell Science 125 (3)
DMEM at 37 ˚C with 5% CO2. 4 mg of each vector construct were transfected using
10 ml Lipofectamine 2000 (Invitrogen) in DMEM without FBS for 24 hours. Then,
medium was changed to normal FBS-containing medium for 1 day and cells were
prepared for further analysis. C2C12 cells (ATCC, Wesel, Germany) were cultured
in the same way as T-Rex293 cells. For differentiation, C2C12 cells were switched
at 70% of confluency to medium containing 2% FBS instead of horse serum.
C57BL/6J mice were used for in vivo BiFC and functional studies. Animals were
from Charles River (Sulzfeld, Germany) and then maintained in the local animal
facility. Use and care of animals was as approved by German authorities and
according to national law (TierSchG§7). An intraperitoneal injection of Rompun
(Bayer, Leverkusen, Germany) and Zoletil 100 (Laboratoires Virbac, Carros
Cedex, France) was used for anesthesia. Transfection was carried out as previously
described (Dona et al., 2003; Röder et al., 2010).
Immunoprecipitation
Journal of Cell Science
After transfection, cells were washed twice with PBS and collected in the presence
of 500 ml lysis buffer (50 mM Tris-Cl pH 8.9, 150 mM NaCl, 1% NP-40, 10%
glycerol, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 1 mM NaF, 0.5 mM
PMSF, 1 mM DTT, 10 mM glycerol phosphate and Complete Protease inhibitor
cocktail). After 30 minutes at 4 ˚C with gentle rocking, lysates were centrifuged
at 13,000 g for 20 minutes and supernatants were incubated with PKA-RIa
monoclonal or rapsyn polyclonal antibody at 4 ˚C in a rotating wheel for overnight.
Then, 50 ml of protein A and protein G agarose mixture (Calbiochem, Darmstadt,
Germany) was added and incubated at 4 ˚C for 2 hours. Finally, samples were
centrifuged for 1 minute at 500 g, washed three times with lysis buffer and eluted
by Laemmli buffer for SDS-PAGE and western blot. For SDS-PAGE, protein
concentrations of every sample were measured using Bradford solution (BioRad,
München, Germany) and further steps were as described (Rudolf et al., 2003).
Cell and tissue staining
After transfection, cells were washed twice with PBS and fixed in 4%
paraformaldehyde at room temperature for 10 minutes. Fixed cells were
permeabilised with 0.1% Triton X-100 at room temperature for 10 minutes and
then quenched with 50 mM NH4Cl. Between each step, cells were washed with
PBS. Blocking was carried out by incubation at room temperature for 1 hour with
10% FBS containing PBS and primary antibody. After washing with PBS,
secondary antibodies and draq5 were added and incubation continued at room
temperature for 1 hour. Finally, cells were washed with PBS and distilled water
and mounted on glass slides with Mowiol. Muscle slicing and immunostaining was
as described (Röder et al., 2010).
Data analysis and statistics
Image analysis was done using the ImageJ program (NIH, Bethesda, MD) or as
described (Röder et al., 2010). Automated NMJ segmentation as used in Fig. 6 and
analysis of AChR turnover and synapse fragmentation were as described [see
figure S5 in Röder et al. (Röder et al., 2010)]. For cellular BiFC analysis,
maximum z-projections of each channel were median-filtered (1 pixel kernel) and
background fluorescence was subtracted. Cells were counted as BiFC-positive if
they exhibited more than 10 pixels above background in the Venus channel.
Stained muscle slices were analysed as follows: first, regions of interest (ROIs)
containing the NMJ area were determined using thresholding of the BGT-AF647positive areas. Then, fibre ROIs containing the entire fibre excluding the NMJ
ROIs were created manually. Mean signal intensities and standard deviations of
PKA-RI or rapsyn antibody staining signals were measured in NMJ and fibre
ROIs. Cumulative plots show the sorted list of values from mean antibody signals
in the NMJ region subtracted by the mean signal plus standard deviation in the
fibre ROIs. Each data point represents one analysed NMJ. All data were tested
for normality using the Kolmogorow–Smirnow–Lilliefors test. Homo- and
heteroscedasticity was evaluated using the F-test. Significance was tested using
either the Student’s t-test or Welch test, as applicable. Values given are
mean 6 s.e.m., unless otherwise stated. Data calculations, statistical analyses
and figure preparation used Microsoft Excel (Unterschleissheim, Germany),
SigmaPlot (San Jose, CA), Adobe Photoshop, and Adobe Illustrator (San Jose, CA)
software.
Acknowledgements
We are grateful to Jonathan Cohen (Boston, MA), Olivier Kassel
(Karlsruhe, Germany) and Veit Witzemann (Heidelberg, Germany)
for generous gifts of constructs. We thank the animal facility of ITG
for their excellent support and Christoph Wilhelm and Susanne
Kaminski (Karlsruhe, Germany) for help with the AChR lifetime
determination.
Funding
R.R. was supported by the Deutsche Forschungsgemeinschaft
[grant number RU923/3-1] and Association Française contre les
Myopathies [grant number 12056].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.092361/-/DC1
Microscopy and AChR lifetime measurements
Imaging and image analysis of live mouse muscles was as described (Röder et al.,
2010). For BiFC experiments using culture cells, images were taken with a DMRE
TCS SP2 confocal microscope equipped with Leica Confocal Software 2.61, a
KrAr laser (488 nm), a diode-pumped laser (561 nm), a HeNe laser (633 nm), and
a 636, 1.4 NA HCX PL APO CS oil immersion objective (Leica Microsystems,
Mannheim, Germany). Fluorescence signals of Venus, mCherry (or AF546) and
draq5 were excited at 488, 561 and 633 nm, and emission was detected using
band-pass settings of 500–550 nm, 570–620 nm, and 650–750 nm, respectively.
3D image stacks were taken at 12-bit and 1024-pixel resolution with 16 or 46
zoom, 200 Hz scan frequency and 26 line average. 125I-BGT-based determination
of AChR turnover was as described (Strack et al., 2011) with the exception that
only about 700 fmol of 125I-BGT was applied per leg.
Computational methods
The profile Hidden Markov model (HMM) of the PKA binding motif of AKAPs
was calculated from AKAP sequence alignment (McLaughlin et al., 2011) and
aligned with the rapsyn fragment using the program HMMER v.3.0 (www.hmmer.
org). The structural model of the complex between PKA-RIa DD domains and
rapsyn was based on the X-ray structure of the complex between PKA-RIa DD
domains and the AKAP10 amphipatic helix solved at resolution 2.29 Å (AKAP10
fragment 628–654, PDB entry code 3IM4) (Sarma et al., 2010). AKAP10 was
replaced by rapsyn fragment 305–331 on the basis of the alignment in Fig. 1A, to
end up with the structural model of the complex between PKA-RIa DD domains
and rapsyn amphipathic helix. The MODELLER program was used to model the
complex (Eswar et al., 2007). Hydrogen atoms were added by assuming standard
bond lengths and angles. The model was immersed in a parallelepiped box whose
edges were 8.3, 8.3 and 8.1 nm, containing 17,765 water molecules; three sodium
ions were added to neutralise the box. The computational setup was as described
(Berrera et al., 2006b). After energy minimisation and 60 picoseconds of solvent
equilibration, the complex underwent 100 nanoseconds of molecular dynamics in
the isothermal-isobaric ensemble at 300 ˚K and 1 atm. The minimum distance
between periodic images of the complex in solution turned out to be always as
large as 3.0 nm. Contact surface, H-bonds and RMSD were calculated as described
(Berrera et al., 2006a).
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